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Identical 3′-termini of the four RNA species of cucumber mosaic virus
VIROLOGY
7+, 853-855
Identical
(1977)
3’-Termini
of the Four RNA Species of Cucumber Virus
YOICHI Central
Research
Institute,
The
TAKANAMI’ Japan
Tobacco Yokohama, Accepted
AND and
SEIKO IMAIZUMI
Salt Public 227, Japan
December
Mosaic
Corporation,
Umegaoka,
Midori-ku,
7,1976
The 3’-ends of the four main RNA species isolated from purified cucumber mosaic virus were labeled with tritium by periodate oxidation, followed by reduction with C3H]borohydride. Each RNA species, fractionated by polyacrylamide gel electrophoresis, was digested with alkali and the resultant 3’-termini were valyzed by phosphocellulose column chromatography. All of the four RNA species have a nonphosphorylated adenosine residue at their 3’-end.
Purified cucumber mosaic virus (CMV) preparations exhibit a single nucleoprotein component in the analytical ultracentrifuge and by sucrose density gradient centrifugation (7, 17). However, RNA preparations extracted from purified CMV show three components by sucrose density gradient centrifugation ( 7) and four major species by polyacrylamide gel electrophoresis (13). The largest RNA species is named component 1; the others are 2, 3, and 4, in turn. It was shown that only RNAs 1, 2, and 3 are needed to produce infection on the leaves of cowpea (10, 13). However, there is little information available about the role of each component, especially about the smallest, component 4. In order to elucidate the structure of the RNA components of CMV, the 3’-terminal nucleoside residues were determined by an end-labeling technique. Fully expanded leaves of tobacco plants (Nicotiuna tubacum cv. Ky57) grown in a greenhouse were inoculated with the yellow strain of CMV (CMV-Y) (18). Three or four days after inoculation, the inoculated leaves were harvested. CMV was purified by essentially the same method as previously described (16). Purified virus suspension was stocked at -80” before use. Virus RNA was obtained from the puri’ To whom
reprint
requests
should
Red CMV preparation by three cycles of deproteinization in the presence of 1% sodium dodecyl sulfate (SDS). The first cycle was carried out at 50” and the others were at room temperature. RNA was precipitated three times by adding ethanol, then dissolved in 0.1 M sodium acetate-O.001 it4 EDTA, pH 5.3. The method used for the 3H-labeling of the 3’-terminal nucleoside was almost the same as that described by Furuichi and Miura (31, involving periodate oxidation and reduction with [3Hlsodium borohydride, except we used 0.2 M sodium borate-0.001 M EDTA-0.1% SDS, pH 8.5, at the reduction step. Polyacrylamide gel electrophoresis of RNA was in 2.4% gel as described by Loening (9). After electrophoresis, the gel was scanned with ultraviolet light and then sliced into 1.6~mm fractions with a multiple razor blade device. Each slice was put into a glass vial and solubilized with 90% NCS (Amersham/Searle), followed by addition of a toluene-based scintillation fluid. Radioactivity was measured in a Beckman LS-200 liquid scintillation spectrometer. An ultraviolet scan of the electrophoresed gel and the radioactivity is shown in Fig. 1. Four main peaks of radioactivity coincided completely with those of ultraviolet absorbance corresponding to the four RNA species. However, several
be addressed. 853
Copyright All rights
0 19’77 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
SHORT
123
4
COMMUNICATIONS
-8 0.6
0.4
0.2 -t L z N 2 0.6 =; L 2 6
d...‘.. 1
2 3 DISTANCE
4 5 MIGRATED
6 7 ( cm)
1. Polyacrylamide gel electrophoresis of the 3H-end-labeled CMV RNA. CMV RNA (20,000 cpm) was applied to a 2.4% gel (0.5 x 7.5~cm column) and subjected to electrophoresis at 5 mA for 2.25 hr. (--I, absorbance at 260 nm; (-----0-----), radioactivity.
0.4 z
FIG.
small peaks of radioactivity besides the main peaks were detected. There were no differences in the pattern of the radioactivity whether RNA was treated with an alkaline phosphatase derived from E. coli (A 19, RNase I-) (4) before labeling or not. A modified method of Furuichi and Miura (3) was employed to analyze the 3’termini of the CMV RNAs. The electrophoresed RNAs in the polyacrylamide gels were stained briefly with 0.05% toluidine blue, and the four main bands were cut out with a razor blade. The gel slices containing RNA were passed through syringe needles attached to plastic syringe barrels. A KOH solution was added to the smashed gels in a test tube to a final concentration of 0.3 N, and the RNAs were hydrolyzed at 37” for 24 hr. Ethanol (70%) was added to the hydrolysates and the supernatants, after low speed centrifugation, were evaporated to dryness under reduced pressure. The resultant residues which contained the 3’terminal nucleoside derivatives of the RNAs released as nucleoside hydroxymethyl diethylene glycols (conveniently called nucleoside trialcohols) were dissolved in water. The gel extract (2 ml) was adsorbed onto an acid-washed charcoal column (0.8 x 0.5 cm). After the column was washed with 2 ml of water, the radioactive nucleoside trialcohols were eluted with 4
0.2
IO
20 30 IO FRACTION NUMBER
20
. 30
FIG. 2. Identification of the 3H-labeled termini of CMV RNA. Terminally labeled CMV RNA species were separated by polyacrylamide gel electrophoresis and hydrolyzed by alkali. After passage through a charcoal column to remove non-nucleoside materials, the sample solutions, to which unlabeled nucleoside trialcohol markers were added, were applied to a phosphocellulose column (0.9 x 10 cm) and eluted with 0.2 M ammonium formate, pH 3.85. Numbers inserted in each figure represent the various CMV RNA species.
ml of 50% ethanol (3). The fractions were evaporated to dryness. The nucleoside derivatives, dissolved in 0.05 M ammonium formate, pH 3.85, were applied to a phosphocellulose column (Whatman P-11, 0.9 x 10 cm) (8) with unlabeled trialcohol markers which were prepared by the method of RajBhandary (14) and eluted with 0.2 M ammonium formate, pH 3.85. Ultraviolet absorption of the added trialcoho1 markers was monitored by an LKB Uvicord at 254 nm. Fractions of 1 ml each were added to dioxane-based scintillation fluid, and their radioactivities were counted. As shown in Fig. 2, most of the radioactivity was detected at the peak of the adenosine-trialcohol fraction with each of the four CMV RNA species. Alkaline phosphatase treatment had no effect on the amount of 3H-incorporation into the 3’-termini; therefore we concluded that they were nonphosphorylated adenosines.
855
SHORT COMMUNICATIONS
When the charcoal column chromatography was omitted after hydrolysis, almost the same number of tritium counts seen on the adenosine-trialcohol marker were observed just prior to the peak of the uridinetrialcohol. These radioactive contaminants, which were ultraviolet absorbant, passed through the phosphocellulose column without being adsorbed. Therefore, they were probably nucleotidic materials derived from the 5’-ends having m7G(5’)ppp(5’)N (15). Single-stranded RNA viruses such as MS2 (21, Qp, f2, R17 (I), tobacco mosaic virus (II ), and brome mosaic virus (BMW (5) have nonphosphorylated adenosine as the 3’4erminal nucleoside of their RNAs. The 3’4ermini of the CMV RNAs are also adenosine. By contrast, RNAs of tobacco rattle virus (TRW (121, tobacco necrosis virus, and its satellite virus (6) have cytidine as their 3’4erminal nucleoside. The oligonucleotide sequence at the 3’-end of the former group is GpCpCpCpAoH and that of the latter is GpCpCpC,, or GpCpCpCp. TRY and BMV, which contain multipartite genomes, have the same 3’-terminal nucleoside in all of their respective RNA species, and our experiments proved the same situation with the CMV RNAs. Recently, Symons (15) reported that the 3’-ends of RNAs extracted from the Q strain of CMV terminated in either adenosine or @dine. The discrepancy between our result and Symons’ might have possibly arisen from the differences in the strain of the virus used or in the analytical methods, although it seems unlikely to us that the 3’4erminus of a species of RNA would terminate in two or more different kinds of nucleosides.
ACKNOWLEDGMENTS We thank Dr. A. Ishihama, Institute of Virus Research, Kyoto University, for his generous gift of E. coli (A19). We also thank Dr. S. Kubo for his comments and suggestions on the preparation of this manuscript. REFERENCES DAHLBERG,
J. E., Nature (London)
220, 548-552
(1968). DEWACHTER, R., and FIERS, W., J. Mol. Biol. 30, 507-527 (1967). FURUICHI, Y., and MIURA, K., Virology 55,418425 (1973). GAREN, A., and LEVINTHAL, C., Biochim. Biophys. Actu. 38, 470-483 (1960). GLITZ, D. G., and EICHLER, D., Biochim. Biophys. Acta 238, 224-232 (1971). HORST, J., FRAENKEL-CONRAT, H., and MANDELES, S., Biochemistry 10, 4148-4752 (19’71). KAPER, J. M., DIENER, T. O., and SCOTT, H. .A., Virology 27, 54-72 (1965). LEWANDOWSKI, L. J., CONTENT, J., and LEPPLA, S. H., J. Viral. 8, 701-707 (1971). 9. LOENING, U. E., Biochem. J. 102, 251-257 (19671.. 10. LOT, H., MARCHOUX, G., MARROU, J., KAPER, J. M., WEST, C. K., VAN VLOTEN-DOTING, L., and HULL, R., J. Gen. Viral. 22, 81-93 (1974). Il. MANDELES, S., J. Biol: Chem. 242, 3103-3107
(1967). 12. 13. 14. 15.
MINSON, T., and DARBY, G., J. Mol. Biol. 77, 337-340 (1973). PEDEN, K. W. C., and SYMONS, R. H., Virology 53, 487-492 (1973). RAJBHANDARY, U. L., J. Biol. Chem. 243, 556564 (1968). SYMONS, R. H., Mol. Biol. Reports 2, 277-285
(1975). Y., and TOMARU, 293-295 (1969). 17. TAKANAMI, Y., and TOMARU, 16.
18.
TAKANAMI,
K.,
Virology
37,
K., Bull. Hatano Tobacco Expt. Sta. 73, 327-332 (1973). TOMARU, K., and HIDAKA, Z., Bull. Hatano Tobacco Expt. Sta. 46, 143-149 (1960).
7+, 853-855
Identical
(1977)
3’-Termini
of the Four RNA Species of Cucumber Virus
YOICHI Central
Research
Institute,
The
TAKANAMI’ Japan
Tobacco Yokohama, Accepted
AND and
SEIKO IMAIZUMI
Salt Public 227, Japan
December
Mosaic
Corporation,
Umegaoka,
Midori-ku,
7,1976
The 3’-ends of the four main RNA species isolated from purified cucumber mosaic virus were labeled with tritium by periodate oxidation, followed by reduction with C3H]borohydride. Each RNA species, fractionated by polyacrylamide gel electrophoresis, was digested with alkali and the resultant 3’-termini were valyzed by phosphocellulose column chromatography. All of the four RNA species have a nonphosphorylated adenosine residue at their 3’-end.
Purified cucumber mosaic virus (CMV) preparations exhibit a single nucleoprotein component in the analytical ultracentrifuge and by sucrose density gradient centrifugation (7, 17). However, RNA preparations extracted from purified CMV show three components by sucrose density gradient centrifugation ( 7) and four major species by polyacrylamide gel electrophoresis (13). The largest RNA species is named component 1; the others are 2, 3, and 4, in turn. It was shown that only RNAs 1, 2, and 3 are needed to produce infection on the leaves of cowpea (10, 13). However, there is little information available about the role of each component, especially about the smallest, component 4. In order to elucidate the structure of the RNA components of CMV, the 3’-terminal nucleoside residues were determined by an end-labeling technique. Fully expanded leaves of tobacco plants (Nicotiuna tubacum cv. Ky57) grown in a greenhouse were inoculated with the yellow strain of CMV (CMV-Y) (18). Three or four days after inoculation, the inoculated leaves were harvested. CMV was purified by essentially the same method as previously described (16). Purified virus suspension was stocked at -80” before use. Virus RNA was obtained from the puri’ To whom
reprint
requests
should
Red CMV preparation by three cycles of deproteinization in the presence of 1% sodium dodecyl sulfate (SDS). The first cycle was carried out at 50” and the others were at room temperature. RNA was precipitated three times by adding ethanol, then dissolved in 0.1 M sodium acetate-O.001 it4 EDTA, pH 5.3. The method used for the 3H-labeling of the 3’-terminal nucleoside was almost the same as that described by Furuichi and Miura (31, involving periodate oxidation and reduction with [3Hlsodium borohydride, except we used 0.2 M sodium borate-0.001 M EDTA-0.1% SDS, pH 8.5, at the reduction step. Polyacrylamide gel electrophoresis of RNA was in 2.4% gel as described by Loening (9). After electrophoresis, the gel was scanned with ultraviolet light and then sliced into 1.6~mm fractions with a multiple razor blade device. Each slice was put into a glass vial and solubilized with 90% NCS (Amersham/Searle), followed by addition of a toluene-based scintillation fluid. Radioactivity was measured in a Beckman LS-200 liquid scintillation spectrometer. An ultraviolet scan of the electrophoresed gel and the radioactivity is shown in Fig. 1. Four main peaks of radioactivity coincided completely with those of ultraviolet absorbance corresponding to the four RNA species. However, several
be addressed. 853
Copyright All rights
0 19’77 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
SHORT
123
4
COMMUNICATIONS
-8 0.6
0.4
0.2 -t L z N 2 0.6 =; L 2 6
d...‘.. 1
2 3 DISTANCE
4 5 MIGRATED
6 7 ( cm)
1. Polyacrylamide gel electrophoresis of the 3H-end-labeled CMV RNA. CMV RNA (20,000 cpm) was applied to a 2.4% gel (0.5 x 7.5~cm column) and subjected to electrophoresis at 5 mA for 2.25 hr. (--I, absorbance at 260 nm; (-----0-----), radioactivity.
0.4 z
FIG.
small peaks of radioactivity besides the main peaks were detected. There were no differences in the pattern of the radioactivity whether RNA was treated with an alkaline phosphatase derived from E. coli (A 19, RNase I-) (4) before labeling or not. A modified method of Furuichi and Miura (3) was employed to analyze the 3’termini of the CMV RNAs. The electrophoresed RNAs in the polyacrylamide gels were stained briefly with 0.05% toluidine blue, and the four main bands were cut out with a razor blade. The gel slices containing RNA were passed through syringe needles attached to plastic syringe barrels. A KOH solution was added to the smashed gels in a test tube to a final concentration of 0.3 N, and the RNAs were hydrolyzed at 37” for 24 hr. Ethanol (70%) was added to the hydrolysates and the supernatants, after low speed centrifugation, were evaporated to dryness under reduced pressure. The resultant residues which contained the 3’terminal nucleoside derivatives of the RNAs released as nucleoside hydroxymethyl diethylene glycols (conveniently called nucleoside trialcohols) were dissolved in water. The gel extract (2 ml) was adsorbed onto an acid-washed charcoal column (0.8 x 0.5 cm). After the column was washed with 2 ml of water, the radioactive nucleoside trialcohols were eluted with 4
0.2
IO
20 30 IO FRACTION NUMBER
20
. 30
FIG. 2. Identification of the 3H-labeled termini of CMV RNA. Terminally labeled CMV RNA species were separated by polyacrylamide gel electrophoresis and hydrolyzed by alkali. After passage through a charcoal column to remove non-nucleoside materials, the sample solutions, to which unlabeled nucleoside trialcohol markers were added, were applied to a phosphocellulose column (0.9 x 10 cm) and eluted with 0.2 M ammonium formate, pH 3.85. Numbers inserted in each figure represent the various CMV RNA species.
ml of 50% ethanol (3). The fractions were evaporated to dryness. The nucleoside derivatives, dissolved in 0.05 M ammonium formate, pH 3.85, were applied to a phosphocellulose column (Whatman P-11, 0.9 x 10 cm) (8) with unlabeled trialcohol markers which were prepared by the method of RajBhandary (14) and eluted with 0.2 M ammonium formate, pH 3.85. Ultraviolet absorption of the added trialcoho1 markers was monitored by an LKB Uvicord at 254 nm. Fractions of 1 ml each were added to dioxane-based scintillation fluid, and their radioactivities were counted. As shown in Fig. 2, most of the radioactivity was detected at the peak of the adenosine-trialcohol fraction with each of the four CMV RNA species. Alkaline phosphatase treatment had no effect on the amount of 3H-incorporation into the 3’-termini; therefore we concluded that they were nonphosphorylated adenosines.
855
SHORT COMMUNICATIONS
When the charcoal column chromatography was omitted after hydrolysis, almost the same number of tritium counts seen on the adenosine-trialcohol marker were observed just prior to the peak of the uridinetrialcohol. These radioactive contaminants, which were ultraviolet absorbant, passed through the phosphocellulose column without being adsorbed. Therefore, they were probably nucleotidic materials derived from the 5’-ends having m7G(5’)ppp(5’)N (15). Single-stranded RNA viruses such as MS2 (21, Qp, f2, R17 (I), tobacco mosaic virus (II ), and brome mosaic virus (BMW (5) have nonphosphorylated adenosine as the 3’4erminal nucleoside of their RNAs. The 3’4ermini of the CMV RNAs are also adenosine. By contrast, RNAs of tobacco rattle virus (TRW (121, tobacco necrosis virus, and its satellite virus (6) have cytidine as their 3’4erminal nucleoside. The oligonucleotide sequence at the 3’-end of the former group is GpCpCpCpAoH and that of the latter is GpCpCpC,, or GpCpCpCp. TRY and BMV, which contain multipartite genomes, have the same 3’-terminal nucleoside in all of their respective RNA species, and our experiments proved the same situation with the CMV RNAs. Recently, Symons (15) reported that the 3’-ends of RNAs extracted from the Q strain of CMV terminated in either adenosine or @dine. The discrepancy between our result and Symons’ might have possibly arisen from the differences in the strain of the virus used or in the analytical methods, although it seems unlikely to us that the 3’4erminus of a species of RNA would terminate in two or more different kinds of nucleosides.
ACKNOWLEDGMENTS We thank Dr. A. Ishihama, Institute of Virus Research, Kyoto University, for his generous gift of E. coli (A19). We also thank Dr. S. Kubo for his comments and suggestions on the preparation of this manuscript. REFERENCES DAHLBERG,
J. E., Nature (London)
220, 548-552
(1968). DEWACHTER, R., and FIERS, W., J. Mol. Biol. 30, 507-527 (1967). FURUICHI, Y., and MIURA, K., Virology 55,418425 (1973). GAREN, A., and LEVINTHAL, C., Biochim. Biophys. Actu. 38, 470-483 (1960). GLITZ, D. G., and EICHLER, D., Biochim. Biophys. Acta 238, 224-232 (1971). HORST, J., FRAENKEL-CONRAT, H., and MANDELES, S., Biochemistry 10, 4148-4752 (19’71). KAPER, J. M., DIENER, T. O., and SCOTT, H. .A., Virology 27, 54-72 (1965). LEWANDOWSKI, L. J., CONTENT, J., and LEPPLA, S. H., J. Viral. 8, 701-707 (1971). 9. LOENING, U. E., Biochem. J. 102, 251-257 (19671.. 10. LOT, H., MARCHOUX, G., MARROU, J., KAPER, J. M., WEST, C. K., VAN VLOTEN-DOTING, L., and HULL, R., J. Gen. Viral. 22, 81-93 (1974). Il. MANDELES, S., J. Biol: Chem. 242, 3103-3107
(1967). 12. 13. 14. 15.
MINSON, T., and DARBY, G., J. Mol. Biol. 77, 337-340 (1973). PEDEN, K. W. C., and SYMONS, R. H., Virology 53, 487-492 (1973). RAJBHANDARY, U. L., J. Biol. Chem. 243, 556564 (1968). SYMONS, R. H., Mol. Biol. Reports 2, 277-285
(1975). Y., and TOMARU, 293-295 (1969). 17. TAKANAMI, Y., and TOMARU, 16.
18.
TAKANAMI,
K.,
Virology
37,
K., Bull. Hatano Tobacco Expt. Sta. 73, 327-332 (1973). TOMARU, K., and HIDAKA, Z., Bull. Hatano Tobacco Expt. Sta. 46, 143-149 (1960).